Synthesis and evaluation of 4-halogeno- N -[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]- N -[11C]methylbenzamide for imaging of metabotropic glutamate 1 receptor in melanoma

Article abstract of DOI:10.1021/jm501845n

Metabotropic glutamate 1 (mGlu1) receptor is found not only in the brain but also in melanomas and breast cancers. mGlu1 is a promising target for molecular imaging-based diagnosis and treatment of melanoma because its overexpression induces melanocyte carcinogenesis. Here we developed three PET tracers: 4-halogeno-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol- 2-yl]-N-[¹¹C]methylbenzamide ([¹¹C]4-6), which exhibited high uptake in target tumor and decreased uptake in nontarget brain tissues. In vitro binding assay indicated high to moderate binding affinities of 4-6 (K_i, 22-143 nM) for mGlu1 receptor. In vivo biodistribution studies in mice implanted with B16F10 melanoma cells confirmed high radioactive uptake in tumor and low uptake in blood, skin, and muscles. Inhibition of mGlu1 receptor using an mGlu1-selective ligand led to reduced radioactive uptake in the tumor. [¹¹C]6 displayed the highest ratio of uptake between tumor and nontarget tissue and may prove useful as a PET tracer for mGlu1 imaging in melanoma.

Full text of DOI:10.1021/jm501845n

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Article
Synthesis and Evaluation of 4-Halogeno-N-[4-[6-
(Isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide
for Imaging of Metabotropic Glutamate 1 Receptor in Melanoma
Masayuki Fujinaga, Lin Xie, Tomoteru Yamasaki, Joji Yui, Yoko Shimoda, Akiko Hatori,
Katsushi Kumata, Yiding Zhang, Nobuki Nengaki, Kazunori Kawamura, and Ming-Rong Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501845n • Publication Date (Web): 20 Jan 2015
Downloaded from http://pubs.acs.org on January 25, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society.
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Journal of Medicinal Chemistry
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Synthesis and Evaluation of 4-Halogeno-
N-[4-[6-(Isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2
-yl]-N-[11C]methylbenzamide for Imaging of
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Metabotropic Glutamate 1 Receptor in Melanoma
Masayuki Fujinaga†,§, Lin Xie†,§, Tomoteru Yamasaki†, Joji Yui†, Yoko Shimoda†, Akiko Hatori†,
Katsushi Kumata†, Yiding Zhang†, Nobuki Nengaki†,‡, Kazunori Kawamura†, and Ming-Rong
Zhang*,†
†Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku,
Chiba 263-8555, Japan; ‡SHI Accelerator Service Co. Ltd., 5-9-11 Kitashinagawa, Shinagawa-ku,
Tokyo 141-8686, Japan
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
§These authors contributed equally
Corresponding Author:
* Ming-Rong Zhang, Phone: +81-43-382-3709, Fax: +81-43-206-3261 E-mail: zhang@nirs.go.jp
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ABSTRACT
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Metabotropic glutamate 1 (mGlu1) receptor is found not only in the brain but also in melanomas
and breast cancers. mGlu1 is a promising target for molecular imaging-based diagnosis and
treatment of melanoma because its overexpression induces melanocyte carcinogenesis. Here, we
developed three PET tracers: 4-halogeno-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-
2-yl]-N-[11C]methylbenzamide ([11C]4–6), which exhibited high uptake in target tumor and
decreased uptake in non-target brain tissues. In vitro binding assay indicated high to moderate
binding affinities of 4–6 (Ki, 22–143 nM) for mGlu1 receptor. In vivo biodistribution studies in mice
implanted with B16F10 melanoma cells confirmed high radioactive uptake in tumor and low uptake
in blood, skin, and muscles. Inhibition of mGlu1 receptor using an mGlu1 selective ligand led to
reduced radioactive uptake in the tumor. [11C]6 displayed the highest ratio of uptake between tumor
and non-target tissue and may prove useful as a PET tracer for mGlu1 imaging in melanoma.
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INTRODUCTION
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Glutamate receptors are divided into iontropic and metabotropic types based on the mechanism by
which their activation gives rise to a postsynaptic current.1,2 Metabotropic glutamate 1 (mGlu1)
receptor, one of eight subtypes of metabotropic type receptors, was originally found to be highly
expressed in the brain.1-3 Followed by brain, mGlu1 was also found ectopically in melanomas,4
breast cancer5 and other cancers6,7. The overexpressed mGlu1 receptor is reported to exhibit
oncogenic characteristics that independently trigger melanocyte tumorigenesis. The melanocytes
show 100% penetrance through continuous activation of mitogen-activated protein kinase and
phoshatidylinositol-3-kinase / protein kinase B pathways in a B-Raf– and N-Ras– independent
fashion.8-11 Contrary to normal skin and benign nevi, which do not express mGlu1, around 68 to
88% of human melanoma biopsy specimens and human melanoma cell lines show overexpression of
mGlu1 receptor.12,13 Further, inhibition or inactivation of mGlu1 was demonstrated to prevent
growth and progression of melanomas. Although a definite mechanistic role of mGlu1 in melanocyte
tumorigenesis is not yet determined, emipirical data suggest that mGlu1 might prove to be a
promising molecular imaging target and may be applied in the diagnosis and personalized treatment
of melanomas.
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Several positron emission tomography (PET) tracers specifically binding to mGlu1 in vivo and
exhibiting high uptake of radioactivity have been developed to visualize mGlu1 receptors in living
brain.14-20 Among these radiotracers, 4-[18F]fluoro-N-[4-(6-(isopropylamino)pyrimidin-4-yl)-
1,3-thiazol-2-yl]-N-methylbenzamide ([18F]FITM, [18F]1, Scheme 1) is a novel PET tracer
displaying high affinity for mGlu1 (IC50: 5.1 nM),21 and is highly selective for human mGlu1 over
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other subtypes (IC50: >7 µM).22,23 PET studies with [18F]1 and its derivatives, [11C]2 and [11C]3,
have been used for qualitative and quantitative investigations on mGlu1 densities and change in
animal models of neurodegenerative diseases.24-28 Moreover, [11C]2 has been used as the first PET
tracer applied for clinical imaging studies of mGlu1 in human brain.29
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Based on the success of various radiotracers in brain imaging, we recently reported the first
experiment on visualization of mGlu1 receptor in melanoma using [18F]1 in PET.30 [18F]1 showed
high uptake of radioactivity in a melanoma model implanted subcutaneously with a mGlu1-positive
B16F10 melanoma cell line. However, owing to a high density of mGlu1 in brain, [18F]1 showed
considerable uptake of radioactivity in the brain, which in turn decreased its selectivity for detection
of tumors over non-targeted organs or tissues and hampered its clinical application.30 Furthermore,
accumulation and slow clearance of radioactivity from brain aroused the possibility of radiation
induced damage. Nevertheless, [18F]1 was considered useful as a lead compound for further
development of radiotracers useful for specific detection of mGlu1-positive melanomas and
noninvasive quantification of mGlu1 expression in tumors.
Scheme 1
To overcome the shortcomings in [18F]1 which undermined its usage in clinical applications, we
designed three novel 11C-labeled radiotracers: 4-chloro ([11C]4), 4-bromo ([11C]5), and 4-iodo
([11C]6)-
N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide
(Scheme 1), by introducing other halogen atoms (chlorine, bromine, or iodine) into [18F]1 instead of
a fluorine atom. The synthesis of [11C]4–6 and the evaluation of their potentials in PET imaging of
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mGlu1 receptors in melanoma formed the aim of this study. It was expected that the replacement of
the smaller fluorine atom by other larger halogen atoms may limit entrance of [11C]4–6 into the brain
while simultaneously maintaining high tumor uptakes. Owing to its short half-life, 11C (half-life:
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20.2 min) is practically inconvenient compared to F (half-life: 109.8 min). We hypothesized that
the use of Br and I in these compounds would render compounds with longer half-lives. It would be
possible to label them using the isotopes of non-standard PET (76Br, half-life: 16 h or 124I, half-life:
4.18 days), SPECT (123I, half-life: 13.27 h) and radiotherapy (131I, half-life: 8 days) without altering
their chemical structures or pharmacological profiles.
In this present study, we report the radiosynthesis of [11C]4–6 and their in vitro binding affinity
for mGlu1, biodistribution, and PET imaging of mice bearing B16F10 melanoma.
RESULTS AND DISCUSSION
Chemical synthesis. Unlabeled target compounds 4–6 and their corresponding desmethyl
precursors 7–9 for radiosynthesis were synthesized according to pathways represented in Scheme 2.
Among these compounds, synthesis of the chlorine analog 4 was reported in a paper previously
published from our laboratory.25 Ethoxyvinylpyrimidine 10 was prepared according to a procedure
described previously.24 Bromination of 10 with 1-bromopyrrolidine-2,5-dione, followed by
treatment with thiourea derivatives, gave 11 and 12 at 83% and 61% yields, respectively.
Benzoylation of 11 and 12 with the appropriate 4-substituted benzoyl chlorides afforded 13–18 at
yields ranging from 19 to 90%. Three precursor compounds 7–9 and targeted compounds 4–6 were
obtained by the reaction of 13–15 and 16–18 with isopropylamine at yields of 26–65%, respectively.
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Scheme 2
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Radiosynthesis of [11C]4–6. Compounds 4–6 were labeled with 11C by N-[11C]methylation of
their corresponding desmethyl precursors 7–9 (Scheme 3). Radiosynthesis was performed using an
automated synthesis system which had been developed in our facility.31,32
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Scheme 3
Radiosynthesis of the chlorine analog [11C]4 was performed by allowing 7 to react with
conventionally used [11C]methyl iodide ([11C]CH3I), which was prepared from the
cyclotron-produced [11C]carbon dioxide ([11C]CO2). However, the radiochemical yield of [11C]4
using [11C]CH3I was low and less than 370 MBq of [11C]4 was obtained by starting with 17 GBq of
[11C]CO2 at the end of synthesis (EOS). This amount of radioactivity was not enough for evaluation
of animal experiments. Thus, to improve the [11C]methylation efficiency, compound 7 was allowed
to react with the more reactive [11C]methyl trifluoromethanesulfonate ([11C]CH3OTf). [11C]CH3OTf
was prepared by passing [11C]CH3I through a heated column coated with silver triflate on
graphitized carbon33 (Scheme 3). The resulting [11C]CH3OTf was subsequently trapped in a solution
of 7 and NaOH in anhydrous acetone at room temperature. Upon completion of reaction, purification
of the reaction mixture by semi-preparative reversed phase HPLC resulted in [11C]4 at radiochemical
yields of 18 ± 5% (n = 7). Similar to [11C]4, [11C]5 or [11C]6 was synthesized by the reaction of 8 or
9 with [11C]CH3OTf. Radiochemical yields were 17 ± 4% (n = 7) or 20 ± 5% (n = 7), respectively.
Radiochemical yields for [11C]4–6 were decay-corrected based on [11C]CO2 at EOS.
Starting from 15.5–22.2 GBq of [11C]CO2, 1.0–1.9 GBq of [11C]4–6 was produced within an
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average synthesis time of 31 min from the end of bombardment (EOB). Specific activity and
radiochemical purity of [11C]4–6 were determined in the final product solution and were 65–160
GBq/µmol and higher than 97% at EOS, respectively. These radioactive products did not undergo
any obvious radiolysis at room temperature until 120 min after formulation, indicating their
radiochemical stability within the duration of at least one PET scan. The analytical results of
[11C]4–6 were in compliance with the quality control and assurance specifications for PET
radiopharmaceuticals produced in our facility.
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In Vitro Binding Assays. The in vitro binding affinity (inhibition constant, Ki) of compounds 4–6
for mGlu1 receptor was measured by a binding assay using the mGlu1-seletive radiotracer [18F]1 in
rat brain homogenates24. As shown in Table 1, compounds 4–6 exhibited either high or moderate
binding affinities for mGlu1, Ki values were observed to be 22, 35, and 143 nM, respectively.
Compared to compound 1 (Ki; 14 nM or 5.4 nM24), the in vitro binding affinity for mGlu1 decreased
with an increase in atomic number in the halogen column. This suggested that the mGlu1 receptor
binding affinity was affected by substitution at the 4-position of the benzene ring in these
compounds. We assume that difference in size or electronic property among the substituted halogen
atoms might have contributed to changed binding affinity of 4–6 for mGlu1.
In the present PET study of melanoma, we used a well established B16F10 melanoma cell line34,35
and C57BL/6J mice implanted with B16F10 cells.30 Previously, immunohistochemistry assays and
in vitro autoradiography could confirm abundance of mGlu1 in mice bearing B16F10.30 The Bmax of
mGlu1 in the B16F10 tumor sections was estimated around 120 fmol/mg protein (120 nM). The
binding affinity of 4–6 for mGlu1 enables their PET imaging of mGlu1 in melanoma, although the
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affinity of 6 seemed insufficient for imaging brain mGlu1 mainly because of lower density of mGlu1
receptor in the brain (for example: 36 nM in rat thalamus26) than that in the melanoma.
Table 1
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Computation and Measurement of Lipophilicity. Theoretically computed lipophilicity values
at pH 7.4 (cLogD) for 4–6 were 2.62–3.42 (Table 1). Following the labeling of these compounds
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with C, lipophilicity (LogD) values of [11C]4–6 were measured using the Shake Flask method.16
The experimentally determined LogD values of [11C]4–6 were 3.04–3.33.
Biodistribution Study in B16F10 Melanoma Mice. The distribution of radioactivity in
mGlu1-positive B16F10 melanoma mice was measured at six experimental time points (1, 5, 15, 30,
60, and 90 min) after the injection of each radiotracer ([11C]4, Table 2; [11C]5, Table 3; and [11C]6,
Table 4).
Table 2
Table 3
Table 4
Radiotracers [11C]4–6 showed similar pharmacokinetic profiles of radioactivity in the major
peripheral organs. High radioactive uptakes (> 5% ID/g) were observed in the blood, lung, heart, and
kidney 1 min after the injection and then decreased rapidly to low levels (<1% ID/g) at 90 min,
respectively. High initial uptakes for [11C]4–6 were also observed in the liver (12.7–15.5% ID/g),
pancreas (3.95–6.15% ID/g), spleen (2.43–2.88% ID/g), and testis (0.58–2.33 % ID/g), and showed
slow decreases until 90 min. Radioactivity showed an increase in the intestine from 1 min and
reached 5.17% ID/g for [11C]4, 3.88% ID/g for [11C]5 and 4.27% ID/g for [11C]6 at 90 min.
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Radioactivity distribution patterns for [11C]4–6 suggested that two pathways, hepatobiliary and
intestinal reuptake, contribute to radioactivity distribution in the whole-body. As expected,
radioactivity levels in blood, muscle, and skin; monitored as control tissues in the present study and
exhibiting low or nil mGlu1 receptor; showed low radioactivity levels until 90 min (0.53–0.85%
ID/g in blood, 0.34–0.46% ID/g in muscle, and 0.59–1.52% ID/g in skin).
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Radioactivity levels increased in the target i.e. tumor tissue immediately upon injecting the
radiotracer and initial uptakes were between 1.71–2.63% ID/g at 1 min. Initial uptake levels
followed the order: [11C]4 > [11C]5 > [11C]6. Radioactivity uptake in the tumor itself increased
gradually after the early phases in case of [11C]4 and [11C]5 and reached the maximum values at 60
min (4.91% ID/g for [11C]4 at 30 min and 4.24% ID/g for [11C]5). Uptake reduced to 3.91% ID/g
and 3.61% ID/g at 90 min, respectively. In case of [11C]6, radioactivity level was higher than 4%
ID/g 15 min after the injection and reached a maximum value of 4.61% ID/g at 90 min. The order of
uptakes in the tumor was [11C]6 > [11C]5 > [11C]4 at 90 min.
mGlu1 is abundantly expressed in the brain leading to a high uptake of the mGlu1 PET
radiotracers. High radioactive uptakes may hinder usefulness of radiotracers in tumor imaging. Thus,
a simultaneous low brain uptake and a high tumor uptake is indispensable for development and
application of a PET tracer for imaging mGlu1 levels in the tumor. In our previous experiments,
[18F]1 was reported to show a high uptake at 90 min not only in the tumor (5% ID/g) but also in the
brain (10% ID/g), limiting its application.30 Compared to [18F]1, [11C]4–6 showed significantly
reduced uptakes and highest radioactivity levels of 5.19% ID/g for [11C]4 at 15 min, 2.97% ID/g for
[11C]5 at 5 min, and 1.65% ID/g for [11C]6 at 5 min after the injection, respectively. Thus, [11C]6
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showed the lowest uptake (0.57% ID/g) in the brain while maintaining the highest uptake (4.67%
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The ratio of radioactivity in tumor and in surrounding regions, i.e. in target regions compared to
healthy non-targeted organs or tissues, is an important index reflective of the specificity of a
radiotracer for the tumor in imaging studies.36 Figure 1 shows the ratios of radioactivity in the tumor
compared to brain, blood, skin, and muscle between 1 and 90 min after the injection of [11C]4–6. All
the four ratios increased with time and [11C]6 showed the highest ratio of tumor to brain, while the
corresponding ratio of tumor to brain for [18F]1 was only 0.5 at 90 min. No significant difference
was observed for [11C]4–6 in the ratios of tumor to blood, tumor to skin, and tumor to muscle,
respectively. This suggested specific accumulation of radioactivity in the tumors. Thus, [11C]4–6
could be further tested for in vivo PET imaging of melanoma in mice owing to their high specificity
for target tissue along with a high degree of portioning of radioactivity in tumors compared to the
background.
PET Study of Mice Bearing B16F10 Melanoma. We performed PET imaging study with
[11C]4–6 for the subcutaneous B16F10-bearing mice. Since the biodistribution study showed
relatively definite radioactivity levels in the tumor between 30 and 90 min after the injection of each
radiotracer (Table 1–3), a time frame between 45 and 75 min was chosen for performing dynamic
PET scans. Images were acquired by summing the uptake of radioactivity within this duration (Figs.
2–4).
Figure 2
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Fig. 2 shows the time-activity curves (TACs) in the tumor and brain of mice between 45 and 75
min after the injection of [11C]4–6. Radioactivity uptake (% ID/g) was similar to biodistribution data
reported above (Table 2–4). These radiotracers displayed similar tumor uptakes (~4.0–4.2% ID/g at
60 min), which were close to previous PET based [18F]1 radioactivity level measurements (4.5%
ID/g) for identical B16F10-bearing mice. Definite radioactive levels of [11C]4–6 were also observed
in the brain. However, values were different from each other. Uptake in brain decreased with an
increase in atomic number of the halogen atom. In particular, the iodo [11C]6 showed much lower
brain uptake than the chloro [11C]4 and bromo [11C]5.
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Figure 3
Fig. 3 summarizes the uptakes of radioactivity, which were quantified using AUC45-75 min (% ID/g
× min), in the mouse tumors and brains. Both [18F]1 and [11C]4–6 showed similar AUC45-75 min
values of ~ 120–130% ID/g in the tumor (p > 0.05). Interestingly, compared to the AUC45-75 min
value in the brain for [18F]1 (380% ID/g ), [11C]4–6 showed significantly reduced AUC45-75 min
values which were 110, 90, and 30% ID/g, respectively. Thus, substitution of fluorine to iodine atom
at position 4 in the benzene ring of [11C]4–6 resulted in limited effects on their uptake, and [11C]6
exhibited the lowest uptake in the brain (p < 0.001).
Figure 4
Representative summed PET images (45–75 min) were acquired under the same scanning
conditions and are shown in Fig. 4. Radioactive signals in the sagittal brain images of control,
particularly in cerebellum and thalamus, decreased in the order: [11C]4 > [11C]5 > [11C]6. High
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amounts of radioactivity was observed to accumulate in the tumor as observed in the horizontal
tumor images of control and low uptake was noted in muscle.
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To determine whether uptake for these radiotracers in the tumor was specific to mGlu1 receptor,
we performed inhibitory experiments using the mGlu1-specific and selective ligand 1. Injection of
excess 1 led to significantly reduced [11C]4–6 radioactivity levels in both the brain and the tumor.
Comparative differences in the TACs (Fig. 2), AUC45-75 min values (Fig. 3), and PET images (Fig. 4)
between the control and treatment with 1 implied specificity of radiotracers for mGlu1 in the tumor.
As shown in the AUC45-75 min values (Fig. 3), the specific binding of [11C]4–6 for mGlu1 accounted
for 51–58% of the total binding. In the brain, about 43% of total binding of [11C]4 and [11C]5 was
specific to mGlu1, whereas no specific binding of [11C]6 was found in the brain. In fact, the brain
uptake of [11C]6 was low.
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This study indicated that substitution of fluorine atom in [18F]1 to iodine atom in [11C]6 could
decrease uptake of radioactivity in brain tissue. This may partially be explained by the lipophilicity
of [11C]6 (LogD: 3.33) along with an decreased binding affinity for mGlu1 receptor compared to
other radiotracers. A high lipophilicity may allow binding of [11C]6 to blood albumin and limit its
entrance into the brain. Although this might also limit the uptake of [11C]6 in the tumor, the effects
of lipophilicity on the brain uptake than on the tumor uptake seem limited. Because the density of
mGlu1 in the brain is lower than that in the melanomas, [11C]6 with its moderate mGlu1 binding
affinity may be able to bind mGlu1 only in the tumor but not in the brain and may selectively bind
melanoma tumors.
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Considering the presence of iodine atom in compound 6 and definite radioactivity level of [11C]6
in melanoma during extended periods, we plan to label 6 with radioactive iodine isotopes 124I and
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131I . This would afford longer half-lives to the radiotracers than those based on C. Prolonging the
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starting time of PET scan after radiotracer injection may be useful for acquiring PET images with
higher signal ratio of tumor to background. More importantly, utilization of the radiotherapy isotope
in the form of 131I-labeled 6 may achieve therapeutic effect for melanoma.
SUMMARY
In summary, three novel radiotracers [11C]4–6 were developed and evaluated for application in
PET-based visualization of mGlu1 receptor in melanoma. [11C]4–6 could be synthesized in a short
synthesis time and reliable radiochemical yields by the N-[11C]methylation of the corresponding
desmethyl precursors 7–9 with [11C]CH3OTf. These PET radiotracers showed high to moderate in
vitro binding affinities for mGlu1 receptor and high uptakes in the melanoma. The ratio of
radioactivity between tumor compared to blood, skin, and muscle was very high. The iodine analog
[11C]6 showed the highest ratio of radioactivity of tumor to brain and is thus regarded as a useful
PET tracer for imaging mGlu1 in melanoma in the future. [11C]6 can be further developed by 124I
and 131I radiolabeling for long-duration PET scan and radiotherapeutic usage. These labeling and
evaluation studies are in progress in our laboratory.
EXPERIMENTAL SECTION
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Materials and Methods. Melting points were measured using a micro melting point apparatus
(MP-500P, Yanaco, Tokyo, Japan) and are uncorrected. 1H–NMR (300 MHz) spectra were recorded
on a JEOL-AL-300 spectrometer (JEOL, Tokyo) using tetramethylsilane (TMS) as an internal
standard. All chemical shifts (δ) are reported as parts per million (ppm) downfield relative to the
TMS signal. Signals are annotated as s (singlet), d (doublet), t (triplet), br (broad), or m (multiplet).
Fast atom bombardment mass spectra (FAB–MS) and high-resolution mass spectra (HRMS) were
recorded on a JEOL-AL-300 spectrometer (JEOL) and recorded on the spectrometer. Silica gel
column chromatography was performed using Wako gel C-200 (70–230 mesh, Wako Pure Chemical
Industries, Osaka, Japan). HPLC analysis was performed using a JASCO HPLC system (JASCO,
Tokyo) and Capcell Pack C18 column (4.6 mm i.d. × 250 mm, Shiseido, Tokyo). Chemical purity of
compounds 4–6 and 7–9 determined by analytical high performance liquid chromatography (HPLC)
was more than 95%. Conditions used for HPLC were as follows: flow rate 1.0 mL/min; solvents,
acetonitrile (MeCN)/H2O/triethylamine (Et3N), (7/3/0.01, v/v/v) for compounds 4–6 and
MeCN/H2O/Et3N, (6/4/0.01, v/v/v) for 7–9.
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Carbon-11 (11C) was produced by a 14N (p, α) 11C nuclear reaction using CYPRIS HM-18 cyclotron
(Sumitomo Heavy Industry, Tokyo). Unless stated otherwise, radioactivity was measured with an
IGC-3R Curiemeter (Aloka, Tokyo). [18F]1 used for the in vitro binding assays was prepared
according to the previously described procedures.22
Chemical synthesis
4-Bromo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide
(5).
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Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 17 (150 mg, 0.37 mmol) and K2CO3
(102 mg, 0.74 mmol) in 1,4-dioxane (5 mL) at room temperature, and the reaction mixture was
heated at 80 °C for 8 h. The reaction mixture was quenched with water and extracted with CH2Cl2.
The organic layer was washed with brine, dried over Na2SO4, and evaporated under reduced pressure.
Residue was purified by column chromatography using n-hexane/ethyl acetate/Et3N (1/1/0.001,
v/v/v) to give 5 (89 mg, 55.6%) as a colorless solid; mp 180–182 °C. 1H NMR (CDCl3): δ 1.29 (6H,
d, J = 6.6 Hz), 3.73 (3H, s), 4.10 (1H, br), 4.84 (1H, br), 7.04 (1H, s), 7.46 (2H, d, J = 8.4 Hz), 7.66
(2H, d, J = 8.4 Hz), 7.93 (1H, s), 8.57 (1H, s). HRMS m/z: 432.0465 (calculated for C18H19ON5BrS:
432.0494).
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4-Iodo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide
(6).
Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 18 (150 mg, 0.33 mmol) and K2CO3
(91 mg, 0.66 mmol) in 1,4-dioxane (12 mL) was added at room temperature. The reaction mixture
was heated at 80 °C for 8 h. The mixture was treated as described for the synthesis of 5. Purification
by column chromatography using n-hexane/ethyl acetate/Et3N (1/1/0.001, v/v/v) gave 6 (103 mg,
65.1%) as a colorless solid; mp 210–211 °C. 1H NMR (CDCl3): δ 1.29 (6H, d, J = 6.2 Hz), 3.73 (3H,
s), 4.12 (1H, br), 4.85 (1H, br), 7.04 (1H, s), 7.32 (2H, d, J = 8.4 Hz), 7.87 (2H, d, J = 8.1 Hz), 7.93
(1H, s), 8.57 (1H, s). HRMS m/z: 480.0386 (calculated for C18H19ON5SI: 480.0355).
4-Chloro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]benzamide
(7).
Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 13 (100 mg, 0.29 mmol) and K2CO3
(79 mg, 0.57 mmol) in 1,4-dioxane (5 mL) was added at room temperature. The reaction mixture
was stirred at 80 °C for 10 h. The mixture was treated as described for the synthesis of 5. Column
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chromatography of the residue on silica gel under CH2Cl2/CH3OH (15/1, v/v) gave 7 (53 mg, 49.7%)
as a white powder; mp 198–200 °C. 1H NMR (DMSO-d6): δ 1.19 (6H, d, J = 6.2 Hz,), 4.13 (1H, br),
6.99 (1H, s), 7.41 (1H, d, J = 7.3 Hz), 7.65 (2H, d, J = 8.4 Hz), 7.92 (1H, s), 8.14 (2H, d, J = 8.4 Hz),
8.42 (1H, s), 12.87 (1H, s). HRMS m/z: 374.0873 (calculated for C17H17ON5ClS: 374.0842).
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4-Bromo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]benzamide
(8).
Isopropylamine (1 mL, 11.7 mmol) was added to a solution of 14 (150 mg, 0.38 mmol) and K2CO3
(105 mg, 0.76 mmol) in 1,4-dioxane (7 mL) at room temperature. The reaction mixture was stirred at
80 °C for 11 h. The mixture was treated as described for the synthesis of 5. Column chromatography
of the residue on silica gel under CH2Cl2/CH3OH (15/1, v/v) gave 8 (93 mg, 58.5%) as a white
powder; mp 202–204 °C. 1H NMR (DMSO-d6): δ 1.19 (6H, d, J = 6.4 Hz), 4.14 (1H, br), 6.99 (1H,
s), 7.41 (1H, d, J = 7.5 Hz), 7.79 (2H, d, J = 8.4 Hz), 7.92 (1H, s), 8.06 (2H, d, J = 8.6 Hz), 8.42 (1H,
s), 12.86 (1H, s). HRMS m/z: 418.0371 (calculated for C17H17ON5BrS: 418.0337).
4-Iodo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]benzamide (9). Isopropylamine
(1 mL, 11.7 mmol) was added to a solution of 15 (182 mg, 0.41 mmol) and K2CO3 (86 mg, 0.62
mmol) in 1,4-dioxane (7 mL) at room temperature. The reaction mixture was stirred at 80 °C for 11
h. The mixture was treated as described for the synthesis of 5. Column chromatography of the
residue on silica gel under CH2Cl2/CH3OH (15/1, v/v) gave 9 (50 mg, 26.3%) as a white powder; mp
201–203 °C. 1H NMR (DMSO-d6): δ 1.19 (6H, d, J = 6.6 Hz), 4.14 (1H, br), 6.99 (1H, s), 7.40 (1H,
d, J = 7.7 Hz), 7.87–7.97 (5H, m), 8.42 (1H, s), 12.84 (1H, s). HRMS m/z: 466.0188 (calculated for
C17H17ON5SI: 466.0199).
4-(6-Chloropyrimidin-4-yl)-thiazol-2-amine (11). 1-Bromopyrrolidine-2,5-dione (2.55 g, 14.3
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mmol) was added to a solution of 4-chloro-6-(1-ethoxyvinyl)pyrimidine24 (10; 2.40 g, 13.0 mmol) in
THF/H2O (40 mL, 1/1) and stirred at room temperature for 2 h. Thiourea (0.99 g, 13.0 mmol) was
added to the reaction mixture and the reaction mixture was stirred for 3 h. The resulting precipitate
was collected by filtration. Moreover, the filtrate was extracted with CH2Cl2. The organic layer was
washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The residue was
purified by column chromatography using n-hexane/ethyl acetate (1/1, v/v) to give 11 (2.3 g, 83.3%)
as a yellow solid; mp. 203–206 °C. 1H NMR (DMSO-d6): δ 7.32 (2H, s), 7.67 (1H, s), 7.76 (1H, s),
8.95 (1H, s). HRMS m/z: 212.9979 (calculated for C7H6N4ClS: 213.0002).
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4-Chloro-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]benzamide
(13).
4-Chlorobenzoyl
chloride (294 µL, 2.3 mmol) was added to a solution of 11 (319 mg, 1.5 mmol) and Et3N (627 µL,
4.5 mmol) in THF (5 mL) under N2 atmosphere. The reaction mixture was stirred at 60 °C for 14 h.
This mixture was quenched with water and extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and evaporated under reduced pressure. Column chromatography of the residue on silica gel
1
under CH2Cl2 gave 13 (100 mg, 19%) as a white powder; mp 245–247 °C. H NMR (DMSO-d6): δ
7.66 (2H, d, J = 8.4 Hz), 8.01 (1H, s), 8.15 (2H, d, J = 8.4 Hz), 8.32 (1H, s), 9.07 (1H, s), 13.00 (1H,
s). HRMS m/z: 350.9884 (calculated for C14H9ON4Cl2S: 350.9874)
4-Bromo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]benzamide
(14).
4-Bromobenzoyl
chloride (241 mg, 1.1 mmol) was added to a solution of 11 (160 mg, 0.75 mmol) and Et3N (314 µL,
2.25 mmol) in THF (5 mL) at room temperature under N2 atmosphere. The reaction mixture was
stirred at 60 °C for 16 h. The mixture was treated as described for the synthesis of 13. Column
chromatography of the residue on silica gel under n-hexane/ethyl acetate (3/1, v/v) gave 14 (188 mg,
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63.5%) as a pale yellow powder; mp 239–241 °C. 1H NMR (DMSO-d6): δ 7.80 (2H, d, J = 8.4 Hz),
8.01 (1H, s), 8.07 (2H, d, J = 8.4 Hz), 8.31 (1H, s), 9.06 (1H, s), 13.00 (1H, s). HRMS m/z: 394.9410
(calculated for C14H9ON4ClBrS: 394.9369).
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4-Iodo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]benzamide (15). 4-Iodobenzoyl chloride
(400 mg, 1.5 mmol) was added to a solution of 11 (213 mg, 1.0 mmol) and Et3N (419 µL, 3.0 mmol)
in THF (5 mL) at room temperature under N2 atmosphere. The reaction mixture was stirred at 60 °C
for 17 h. The mixture was treated as described for the synthesis of 13. Column chromatography of
the residue on silica gel under n-hexane/ethyl acetate (3/1, v/v) gave 15 (340 mg, 76.8%) as a white
powder. mp 227–229 °C. 1H NMR (DMSO-d6): δ 7.90 (2H, d, J = 8.4 Hz), 7.97 (2H, d, J = 8.4 Hz),
8.01 (1H, s), 8.31 (1H, s), 9.07 (1H, s), 12.98 (1H, s). HRMS m/z: 442.9267 (calculated for
C14H9ON4ClSI: 442.9230)
4-Bromo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide
(17).
4-Bromobenzoyl chloride (1.65 g, 7.5 mmol) was added to a solution of 12 (1.14 g, 5.0 mmol) and
Et3N (2.1 mL, 15.0 mmol) in toluene (30 mL) at room temperature under N2 atmosphere. The
reaction mixture was stirred at 100 °C for 6 h. This mixture was quenched with water and extracted
with CH2Cl2. The organic layer was dried over Na2SO4 and evaporated under reduced pressure.
Column chromatography of the residue on silica gel under CH2Cl2/Et3N (1/0.001, v/v) and then
CH2Cl2/ethyl acetate (10/1, v/v) gave 17 (1.1 g, 54%) as a white powder; mp 225–226 °C. 1H–NMR
(CDCl3): δ 3.76 (3H, s), 7.48 (2H, d, J = 8.4 Hz), 7.68 (2H, d, J = 8.4 Hz), 8.06 (1H, s), 8.13 (1H, s),
8.95 (1H, s). HRMS m/z: 408.9545 (calculated for C15H11ON4ClBrS: 408.9525).
4-Iodo-N-[4-(6-chloropyrimidin-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (18). 4-Iodobenzoyl
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chloride (264 mg, 0.99 mmol) was added to a solution of 12 (150 mg, 0.66 mmol) and Et3N (275 µL,
2.0 mmol) in toluene (5 mL) at room temperature under N2 atmosphere. The reaction mixture was
stirred at 100 °C for 6 h. The mixture was treated as described for the synthesis of 17. Column
chromatography of the residue on silica gel under CH2Cl2/Et3N (1/0.001, v/v) and then CH2Cl2/ethyl
acetate (10/1, v/v) gave 18 (210 mg, 70.0%) as a white powder; mp 281–282 °C. 1H–NMR (CDCl3):
δ 3.75 (3H, s), 7.34 (2H, d, J = 8.6 Hz), 7.89 (2H, d, J = 8.6 Hz), 8.05 (1H, s), 8.13 (1H, s), 8.95 (1H,
s). HRMS m/z: 456.9354 (calculated for C15H11ON4ClSI: 456.9387).
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Radiochemistry
4-Chloro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide
([11C]4). [11C]CO2 produced in the cyclotron after irradiation was bubbled into 0.4 M lithium
aluminum hydride in anhydrous tetrahydrofuran (THF, 0.3 mL). After evaporation of THF, the
remaining complex was treated with 57% hydroiodic acid (0.3 mL) to give [11C]CH3I, which was
distilled and transferred through a glass tube containing sliver trifluoromethanesufonate and
graphitized carbon under N2 gas flow to yield [11C]methyl trifluoromethanesulfonate ([11C]CH3OTf).
[11C]CH3OTf was immediately transferred into a solution containing 7 (1.0 mg) and 0.5 M aqueous
NaOH solution (5 µL) in anhydrous acetone (300 µL) at room temperature. The reaction mixture
was kept at room temperature for 5 min after the radioactivity reached a plateau. After removal of
acetone, 1.0 mL of the preparative HPLC mobile phase was added. HPLC separation was completed
on a Capcell Pack C18 column (10 mm i.d. × 250 mm; Shiseido) using MeCN/H2O/Et3N (7/3/0.01,
v/v/v) at 4.5 mL/min. The radioactive fraction corresponding to [11C]4 (tR: 7.7 min) was collected in
a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and
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passed through a 0.22 µm Millipore filter to give 1.9 GBq of [11C]4. The identity of [11C]4 (tR: 5.8
min) was confirmed by analytical HPLC with 4. Specific activity was calculated by comparing the
assayed radioactivity to the mass measured at UV 254 nm. The synthesis time was 30 min from
EOB; radiochemical yield (decay-corrected), 20% based on [11C]CO2; radiochemical purity, > 99%;
specific activity at EOS, 130 GBq/µmol.
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4-Bromo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide
([11C]5). [11C]CH3OTf was transferred immediately upon synthesis into a solution containing 8 (1.0
mg) and 0.5 M aqueous NaOH solution (5 µL) in anhydrous acetone (300 µL) at room temperature.
The reaction mixture was kept further for 5 min at room temperature after radioactivity reached a
plateau. After removal of acetone, 1.0 mL of the preparative HPLC mobile phase was added. HPLC
separation was completed on a Capcell Pack C18 column (10 mm i.d. × 250 mm; Shiseido) using
MeCN/H2O/Et3N (7/3/0.01, v/v/v) at 4.5 mL/min. The radioactive fraction corresponding to [11C]5
(tR: 8.1 min) was collected in a sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of
sterile normal saline, and passed through a 0.22 µm Millipore filter to give 1.4 GBq of [11C]5. The
identity of [11C]5 (tR: 6.3 min) was confirmed by analytical HPLC using 5. Specific activity was
calculated by comparing the observed radioactivity with the mass measured at UV 254 nm. The
synthesis time was 31 min from EOB; radiochemical yield (decay-corrected), 23% based on
[11C]CO2; radiochemical purity, > 99%; specific activity at EOS, 160 GBq/µmol.
4-Iodo-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide
([11C]6). The resulted [11C]CH3OTf and transferred under N2 gas flow into a solution containing 9
(1.0 mg) and 0.5 M aqueous NaOH solution (5 µL) in anhydrous acetone (300 µL) at room
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temperature. The reaction mixture was kept at room temperature for 5 min after the radioactivity
reached a plateau. After removal of acetone, 1.0 mL of the preparative HPLC mobile phase was
added, the final mixture was applied to the HPLC system. HPLC separation was completed on a
Capcell Pack C18 column (10 mm i.d. × 250 mm; Shiseido) using MeCN/H2O/Et3N (7/3/0.01, v/v/v)
at 4.5 mL/min. The radioactive fraction corresponding to [11C]6 (tR: 8.5 min) was collected in a
sterile flask, evaporated to dryness in vacuo, redissolved in 3 mL of sterile normal saline, and passed
through a 0.22 µm Millipore filter to give 1.8 GBq of [11C]6. The identity of [11C]6 (tR: 7.1 min) was
confirmed by analytical HPLC with 6. The synthesis time was averaged to be 31 min from EOB;
radiochemical yield (decay-corrected), 25% based on [11C]CO2; radiochemical purity, > 99%;
specific activity at EOS, 100 GBq/µmol.
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Computation and Measurement of Lipophilicity. The cLogD values of compounds 4–6 were
determined computationally using Pallas 3.4 software (CompuDrug, Sedona, AZ). The LogD value
was measured by mixing [11C]4–6 (radiochemical purity: 100%; about 200,000 cpm) with n-octanol
(3.0 g) and phosphate buffered saline (PBS; 3.0 g, 0.1 M, pH 7.4) in a test tube. After vortexing for 3
min at room temperature, contents were centrifuged at 2,330 g for 5 min. An aliquot of 1 mL PBS
and 1 mL n-octanol was removed, weighed, and its radioactivity was counted using a 1480 Wizard
autogamma counter (Perkin-Elmer, Waltham, MA), respectively. Each sample from the remaining
organic layer was removed and repartitioned until a consistent LogD value was obtained. The LogD
value was calculated by comparing the ratio of cpm/g of n-octanol to that of PBS and expressed as
LogD = Log[cpm/g (n-octanol)/cpm/g (PBS)]. All measurements were performed in triplicate.
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Animal Experiments. Animal studies were approved by the Animal Ethics Committee of
National Institute of Radiological Sciences (Chiba. Japan). The animals were maintained and
handled in accordance with the recommendations of National Institute of Health and the institutional
guidelines of National Institute of Radiological Sciences.
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In Vitro Binding Affinity for mGlu1 Receptor. Sprague-Dawley rats (8 week, 250–290 g, n =
5) were sacrificed by decapitation under anesthesia with isoflurane (5 % in air). Entire rat brains
were rapidly removed after sacrifice and homogenized in 10 volumes of 50 mM Tris-HCl (pH, 7.4)
containing 120 mM NaCl with a Silent Crusher S homogenizer (Heidolph Instruments). The
homogenate was centrifuged in polypropylene tube at 40,000 g for 15 min at 4°C using an
Optima-TLX (Beckman Coulter). Supernatant and the pellet was resuspended and centrifuged in the
same buffer. This procedure was repeated twice and the final brain homogenate pellet was stored at
−80°C until further experimental use.
In vitro binding assay to measure affinity for mGlu1 receptor using brain homogenate was
performed according to a method described elsewhere.24 Briefly, the brain homogenate was
incubated with [18F]1 (3 nM in PBS) and multi-dose tested compounds (10−6 to 10−10 M in 0.1%
dimethylsulfoxide). The results of inhibitory experiments were subjected to nonlinear regression
analysis using Prism 5 (GraphPad Software, La Jolla, CA) by which IC50 and the inhibition constant
(Ki) values were calculated.
Preparation of Tumor Model Mice. Melanoma B16F10 cell line (American Type Culture
Collection, Manassas, VA) was maintained and passaged in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum, penicillin (100 unit/mL) and streptomycin
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(0.1 mg/mL). Animal experiments were performed in 6–8-week-old male C57BL/6J mice (Japan
SLC, Shizuoka, Japan). Subcutaneous tumor-bearing models were created via injection into the left
flank with a single-cell suspension of 1 × 106 B16F10 cells in 100 µL DMEM medium without
serum. Mice with a maximum tumor diameter of 9–12 mm were selected for evaluation studies 10
days after subcutaneous inoculation.
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Biodistribution Studies. The B16F10-bearing mice were sacrificed by cervical dislocation at
designated time points after injection of [11C]4–6 (1.70 to 1.85 MBq/0.1 mL). Tumors and major
organs and tissues (blood, lung, heart, kidney, liver, pancreas, spleen, testis, intestine, muscle, and
skin, brain), were promptly excised, harvested, and weighed. Radioactivity was counted using a
g-counter and expressed as a percentage of the injected dose per gram of wet tissue (% ID/g). All
radioactivity measurements were corrected for decay. The ratios of tumor-to-blood, tumor-to-muscle,
and tumor-to-skin were calculated.
Small-animal PET Scans. PET scans were performed for B16F10-bearing mice using a
small-animal Inveon PET scanner (Siemens, Knoxville, TN) after intravenous injection of [11C]4–6
(12–17 MBq/0.1 mL). The scans provided 159 transaxial slices with 0.796 mm (center-to-center)
spacing, a 10 cm transaxial field of view (FOV), and a 12.7 cm axial FOV. Emission scans were
acquired in a 3-dimensional list mode with an energy window of 350–650 keV under isoflurane
anesthesia starting from 45 to 75 min after injection in the subcutaneous tumor models. Blocking
experiments for [11C]4–6 uptake were performed by injecting excess unlabeled 1 (10 mg/kg) at 1
min before radiotracer injection, to inhibit the binding of radiotracers to mGlu1 receptor. All
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list-mode acquisition data were sorted into 3-dimensional sinograms, which were then
Fourier-rebinned into 2-dimensional sinograms and corrected for scanner dead time, randoms, and
decay of the injected radiotracer. Dynamic images were reconstructed with filtered back-projection
using a Hanning’s filter and a Nyquist cutoff of 0.5 cycles/pixel. Regions of interest in the tumors
and right flank muscles were drawn using the Siemens Inveon Research Workplace (IRW) 4.0
software. The average radioactivity concentration in the tumors and tissues was obtained from mean
pixel values in the region of interest volume, which was manually positioned based on the tumor and
tissue contour in the different orthogonal planes with the largest diameter. Regional uptake of
radioactivity was decay-corrected to injection time, normalized to weights, and expressed as % ID/g.
Time-activity curves (TACs) of [11C]4–6 were determined. Values of area under time-activity curves
(AUC45–75 min, % ID/g × min) in the subcutaneous tumors were calculated.
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Statistical analysis. All data are presented as the mean of the values ± the standard error of mean
(SEM). Comparisons of brain or tumor uptake among [11C]4–6 were performed using one-way
ANOVA test. The threshold for statistical significance was set at p < 0.05. All in vivo experimental
results included data from 4–6 mice in the respective groups, and all in vitro experimental results are
representative of 3 independent experiments and indicated as the mean ratio of triplicate results in
each experiment.
ASSOCIATED CONTENT
Supporting Information Available. Purities of compounds 4–6 and 7–9 determined by HPLC,
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HPLC analytic charts of 4–9, HPLC purification charts for [11C]4–6; HPLC analysis charts for
[11C]4–6. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author
* Phone: +81-43-382-3709; Fax: +81-43-206-3261; E-mail: zhang@nirs.go.jp. Address: Molecular
Probe Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1
Anagawa, Inage-ku, Chiba 263-8555, Japan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We would like to thank the staff at the National Institute of Radiological Sciences for their support
with the cyclotron operation, radioisotope production, radiosynthesis, and animal experiments. This
study was supported in part by Grants-in-Aid for Scientific Research (Basic Research B: 22591379)
from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese
Government.
ABBREVIATIONS USED
AUC, area under the time-curve; [11C]CH3I, [11C]methyl iodide; [11C]CH3OTf, [11C]methyl
trifluoromethanesulfonate, [11C]CO2, [11C]carbon dioxide; EOB, end of bombardment; EOS, end of
synthesis; Et3N, triethylamine; FAB-MS, fast atom bombardment mass spectra; HPLC, high
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performance liquid chromatography; HRMS, high resolution mass spectra; % ID/g, percentage of the
injected dose per gram of wet tissue; Ki, inhibition constant; MeCN, acetonitrile; mGlu1,
metabotropic glutamate 1 receptor; PBS, phosphate buffered saline; PET, positron emission
tomography; SUV, standardized uptake value; TAC, time-activity curve; tR, retention time.
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